This invention relates to a complex for oral delivery of drugs, therapeutic protein/peptides, vaccines, which are loaded in Vitamin B12 coupled particulate carriers system with spacers in between. Typically, VB12 acts as ligand for endocytosis through intestine and the particulate systems will act as a cargo protecting the intestinal labile drugs to deliver them to systemic destination and/or to the specific site required.
Although many peptide/proteins, pharmaceuticals and vaccines are currently administered by injection, this method of delivery has a number of disadvantages which has led the scientific community to strive to develop an alternative oral delivery system for these bioactives. The inherent limitations to parenteral delivery include (1) patient compliance (there is a need for repeated injections due to the short half-life of these molecules) (2) discomfort from this form of delivery caused by the need for repeated prolonged dosage regimen (3) highly variable bioavailability both within and between subjects for molecules such as subcutaneous insulin and (4) the non-physiological delivery pattern particularly of subcutaneous injections. Further, small increases in actual drug delivered due to changes in dosage and/or mode of delivery may cause down-regulation of the desired response to most of these bioactives. In contrast most often a pulsatile or flat delivery profile is required which mimics the normal physiological rhythm. Apart from the problem described, parenteral vaccines are of limited efficacy due to the need for repeated vaccination and due to the fact that they elicit only humoral immunity.
To address the above problems, various non-invasive delivery systems have been attempted. The major delivery barriers common to these routes are (1) poor intrinsic permeability due to large size and hydrophilicity of the bioactive (2) enzymatic degradation in the hostile environment of GIT by lumenal proteases and cellular peptidases, unlike some of the traditional drugs. Although these odds are formidable for the per oral route, it enjoys advantages in terms of convenience and patient compliance as well as safety, less stringent quality control, cost of therapy and for vaccines, the potential for unlimited frequency of boosting. Furthermore, oral vaccines offer the potential to protect against not only enteric pathogens (by producing localized sIgA), but also a wide range of pathogens infecting other mucosa (respiratory and genital) by producing a common disseminated mucosal immune response. Furthermore oral vaccines may prove particularly useful in the elderly because mucosal immunity, unlike systemic immunity does not seem to be an age associated dysfunction. This mode of immunization may also be beneficial in the very young, because mucosal immunity develops earlier in ontogeny than systemic immunity. Over the past few decades significant efforts have been made to develop oral protein/vaccine systems using permeation enhancers (ZOT, which has proven to be toxic on long term use), enzyme inhibitors, surfactants, emulsions, colon targeted systems, bioadhesive systems and particulate systems. Although the challenges to develop oral systems are numerous, the potential therapeutic need remains high, particularly with increased identification of novel peptides and increased production of existing protein drugs from the biotechnology arena.
Peptide/protein drugs and vaccines are mostly given by parenteral routes. The problems for the oral delivery are:
1) Degradation of the above bioactives in the harsh environment of intestine and by gut proteases.
2) Their large size and hydrophilicity causes permeability problem across the intestine.
3) The fragility of these bioactives precludes to be formulated as oral dosage forms.
4) And finally their short in-vivo half lives.
Over the past 3-4 decades efforts have been made to deliver them by oral route using various formulation approaches such as emulsions, microspheres, nanoparticles, vasicular carriers such liposomes, using permeation enhancers and protease inhibitors and by protein-carrier conjugates.
Recently it has been shown that the problem of poor intrinsic permeability can be possibly overcome by delivery via a specific carrier mechanism that transports pharmaceuticals from the intestine into the circulation. In this regard, Russell-Jones and co workers [1994] have found the possibility of coupling these bioactives with Vitamin B12 (VB12) in a manner that does not interfere with intrinsic factor (IF) mediated uptake of the VB12 carrier to the systemic circulation. Some interesting results have been achieved with oral VB12 conjugates of LHRH (Russell-Jones 1998), interferon (Habberfield and Jensen-Pippo PPO, 1996) G-CSF and erythropoietin (Habberfield, 1996). However, such successful oral delivery of conjugates cannot be obtained with many other bioactives because of the limited uptake of VB12 (1 nmol/dose), the loss of bioactivity due to covalent linkage, loss of IF affinity of VB12 (steric factor) and finally the liability of such conjugates to GIT degradation. To address the above problems we have made endeavors to make an oral delivery system (VB12-sphere conjugate) that could be targeted to the systemic circulation through VB12-IF-IFR ligand-mediated endocytosis via ileocytes of the intestine following oral administration. In vitro transport of VB12 coated polystyrene nanoparticles (non-biodegradable, hydrophobic) has been demonstrated across Caco-2 cell cultures (Russell Jones, 1997, 1999). However, the level of transport cannot be extended to other polymeric particulates in general. This can be explained by the differing physicochemical characteristics of polymers such as hydrophobicity, as hydrophobic materials may be more readily taken up by cell surface lipid bilayers. Also one of the prerequisites for drug delivery is that the polymer used to make the nanoparticles should readily or slowly be degraded in the systemic environment or by enzymes which release the pharmaceutical molecule and thereby initiate its bioactive response. Hence, the use of nonbiodegradable polymers for transport studies is not necessarily indicative, of uptake by other biodegradable polymers in general. Further, developing a system of loading of hydrophilic peptide/protein or vaccine bioactives into hydrophobic polymeric carriers is definitely not obvious.
Among all these approaches, there was some enhancement of delivery, but major break through of achieving therapeutically relevant dose delivery is not achieved. In the recent past, a delivery system was invented by one of the co-inventors using VB12 as carrier molecule which is coupled with protein drug to be delivered i.e. VB12-protein conjugates. VB12 binds with intrinsic factor of the intestine (VB12+IFxe2x86x92VB12 IF) in the duodenum and the whole VB12 IF complex binds to intrinsic factor receptor (IFR) at the ileum of the small intestine. From there, it is endocytosed and subsequently transcytosed by transcobolamin (TcII) to reach systemic circulation.
Therefore, if protein is coupled with VB12, it will be co-transported along with VB12 molecule to reach systemic circulation.
Two conditions are to be satisfied for this approach i.e.
Case 1: VB12 molecule must retain its IF affinity after being coupled to protein to be delivered.
Case 2: Also protein molecule must retain its bioactivity after coupling to VB12.
For case 1: Native VB12 (cynocobolainin) cannot be directly coupled to protein, i.e. VB12 losses its IF affinity by such coupling.
Therefore, in the above technology VB12 is hydrolyzed, where it forms 3 isomers (a, b and e isomers). Among the 3 isomers, the xe2x80x98exe2x80x99 isomer retains considerable IF affinity after coupling to other molecules.
Case 2: For the ease of coupling to provide suitable groups and to retain full bioactivity of protein drug, various derivatives of e-VB12 using various spacers. These derivatives are coupled to protein drugs to be delivered. Such spacers also increases IF affinity of e isomer. About this technology, one of the inventors of the present application namely Dr. Gregory Russell Jones written a chapter in a book in 1995 (peptide based drug design Chapter 8). In this chapter, the author elaborated this technology and summarized the results of VB-LHRH analogs and VB12 vasopressin and also mentioned the possibility of conjugating other bioactives such as EPO and G-CSF. Later in 1995, two research papers appeared in bioconjugate chemistry (1) VB12-LHRH analogs (Volume 6, No. 1, 1995, 34-42), (2) VB12-EPO and VB12-G CSF (Volume 6, No.4, 1995, 461-465). Later in 1996, one of the authors patented VB12-EPO and VB12-G CSF conjugates (U.S. Pat. No. 5,480,68, August 1998. In this patent he claimed any derivative of VB12 which retains IF affinity after being coupled protein drug to be delivered. Later, in November 1996 there is another patent (U.S. Pat. No. 5,574,018) on the similar system (Habber Field, who is associated D. Russell on some research paper works earlier) i.e. VB12-EPO, VB12-G SCF and VB12-Interferon. But the derivative used is different i.e. at hydroxy site of ribose of VB12. This derivatization at this site and derivatives coupled to protein drug also retain IF affinity of VB12. But in the earlier patent any derivative of VB12, which retains IF affinity after being coupled to protein drugs is claimed. Protein drugs used in the other patent are also same with exception of interferon.
Two U.S. Pat. Nos. 5,589,463 and 5,807,832 wherein, the one of the coinventor of the present application is also main inventor describe VB12-bioactive chemical (covalent) conjugate. The bioactive may be protein, vaccine or antigen (may be of polysaccharide nature) or other biopharmaceutical (heparin) or traditional drug (neomycin). The above chemical complexes they claim to formulate in known dosage forms suitable for oral delivery gels, dispersions, emulsions, capsules, tablets etc. Technology of both these patents is somewhat similar to other patents, U.S. Pat. Nos. 5,548,064 and 5,574,018, which are, referred above.
In spite of all these, the conjugates of VB12 have the following drawbacks.
1) Protein is not protected from degradation by proteases in gut.
2) All proteins cannot be coupled, as some protein bioactives may loose active molecular confirmation due to its coupling to bulky VB12 (steric factors).
3) Uptake of VB12 transport system (1 nano gram g/dose) is not sufficient for proteins which have very short half lives i.e. therapeutically relevant dose cannot be achieved.
The present invention is entirely different in that bioactive (protein/vaccine or drug) is not directly coupled to VB12 or its analogue. Instead, VB12 or its analogues are covalently coupled to micro/nano particle surface in which bioactive is loaded. The micro and nano particles are made up of biodegradable and pharmaceutically acceptable polymers.
The conjugation of various peptides and proteins to the Vitamin B12 molecule has been shown to facilitate the in-vitro and in-vivo transport of these moieties across the epithelial cells of the intestine. However, pharmaceutically relevant oral delivery of many vitamin B12-pharmaceutical conjugates does not occur with many of these bioactives due to the limited uptake capacity of the VB12 transport system, loss of bioactivity of native protein during conjugation to VB12, loss of intrinsic factor (IF) affinity of the conjugates and finally due to the liability of the bioactives to GI degradation. In order to overcome these shortcomings we have endeavoured to develop vitamin B12-particulate (both microspheres and nanospheres) systems that could be taken up by the natural uptake mechanism for VB12. Different biodegradable particulate systems (microspheres and nanospheres) were prepared. These particles were surface modified with vitamin B12 and these preformed sphere conjugates were loaded with therapeutic proteins (insulin, hepatitis xe2x80x98Bxe2x80x99 vaccine). In some cases the loading step was altered. In-vitro studies showed that insulin was protected from degradation by gastric enzymes. These systems, of different sizes, were fed to diabetic rats, whose blood glucose levels were monitored over time. The pharmacological bioavailability of these systems was 5-25%, which showed not only oral bioactivity but also evidence of controlled release of these biopharmaceuticals. These systems showed interesting results with vaccine loaded VB12 microspheres and nanospheres. Thus the above carrier system and various strategies mentioned herein, upon further testing and optimization, may make per oral protein delivery a reality.
The present invention relates to the field of pharmaceutical preparation of traditional injectable drugs given parenterally, peptide and protein pharmaceuticals including vaccines, particularly in the field of such pharmaceutical preparations, which are suitable for oral delivery.
In accordance with the present invention there is provided a drug delivery system comprising a protein drug (insulin and hepatitis xe2x80x98Bxe2x80x99 vaccine) incorporated within the biodegradable polymeric particulates, which are coated with VB12 on their surface as shown in FIG. 1, wherein D is the injectable drug, SP- is the long chain spacer to retain IF affinity of Vitamin B12 and/or for ease of coupling, BDC is the biodegradable carrier particle (microsphere/nano particle).
For the purpose of successful oral drug delivery, VB12 acts as targeting ligand for initial transport across the intestinal epithelium the circulation or for site-specific delivery of the vaccines. The VB12 carries the particulate spheres (micro and nano) along with it until the desired site reached.
In the embodiment of the present invention, VB12-microsphere and VB12-nanoparticle systems deliver the traditional parenteral drugs, peptides/proteins including vaccines by oral route. Various biodegradable microspheres and nanoparticies are prepared with existing natural, semi synthetic and synthetic polymers. These particulates systems (micro and nano) can be surface modified to provide groups suitable for VB12 conjugation.
Vitamin B12 derivatives or their various derivatives with numerous spacers (Russell Jones et al 1995a and 1995b) were coupled to particulate carriers. Different linkages, both biodegradable (cleaved by systemic enzymes) and non-biodegradable covalent linkages are used to link the VB12 to the particulate. The coupling also involves ionic, coordinate covalent. and strong physical adsorptive bounds.
In an embodiment, the drugs to be delivered are the traditional drugs administered exclusively by parenteral routes, therapeutic peptides/proteins, other biopharmaceuticals such as heparin and vaccines for immunization.
In another embodiment of the present invention, the drugs to be delivered are the traditional drugs administered exclusively by parenteral routes selected from gentamycin and Amikacin, therapeutic peptides/proteins selected from insulin, EPO, G-CSF, GM-CSF, Factor VIR, LHRH analogues and Interferons, other biopharmaceuticals such as heparin and vaccines selected from Hepatitis xe2x80x98Bxe2x80x99 surface antigen, typhoid and cholera vaccine for immunization.
In yet another embodiment, the carrier system is a pharmaceutically acceptable carrier comprising VB12 or its analogs or their derivatives to be coupled to biodegradable polymeric particulate carriers, which are, loaded with drugs/vaccines given parentally.
In yet another embodiment, the carrier system is transcytosed after receptor mediated endocytosis at or around ileum by the natural uptake VB12.
In yet another embodiment, VB12 is the targeting ligand for the intestine to systemic and/or lymphatic destiny.
In yet another embodiment, VB12 is native cynocobalamine (VB12) or various analogs of VB12 viz., aquocobalimin, adenosylcobalamin, methylcobalamin, hydroxycobalamin and/or their derivatives or alkylcobalamines in which alkyl chain is linked to cobalt of VB12 or cynocobolamin with chloro, sulphate, nitro, thio or their analide, ethylamide, propionamide, monocarboxylic and dicarboxylic acid derivatives of VB12 and its analogues or monocarboxylic, dicarboxylic and tricarboxylic acid derivatives and prominamide derivatives of xe2x80x98exe2x80x99 isomer of monocarboxy VB12 and analogues of VB12 in which cobalt is replaced by other metals (zinc or nickel etc) and various spacers attached to these derivatives or any derivatives which retains IF affinity after coupling to biodegradable particulate carriers.
In yet another embodiment, the derivatives of VB12 also include derivatization at primary 5xe2x80x2-hydroxyl and 2-hydroxyl groups of ribose moiety of VB12 and various spacers attached at this site useful for coupling with particulate carriers.
In yet another embodiment, Rxe2x80x2-Rxe2x80x3- the spacers and/or agents for derivatization of VB12 to provide either NH2 or COOH or SH groups and combination thereof are selected from diacids or alkyl diacids (COOHxe2x80x94COOH, (COO(CH2)nCOO)) alkyl diamines (NH2(CH2)nxe2x80x94NH2) or alkyl diamides (NHCO (CH)n CONH2) or hydrazides (NH2NH2) or alkyl dihydrazides (NH2NHCO(CH2)n CONHNH2 or substitution SH group containing agents (N-succinymnidyl3-(2-pyridyldithio)propionate or its long chain alkyl derivatives or acid anhydrides [(CH)n COCOO)] or acid halide spacers (R(CH2)n COCl) or anhydroxy activated ester functional groups (NHS) which are used for the peptide bonds or surfactant derivatives or polymeric spacers, and in all cases xe2x80x98nxe2x80x99 is an integer from 1,2,3 . . . infinite.
In yet another embodiment, the microspheres or nanoparticles are made up of biodegradable and pharmaceutically acceptable polymers.
In yet another embodiment, the drug loaded polymeric particulates degrade in-vivo to release and deliver a protein pharmaceutical/vaccine for its bioactive response.
In yet another embodiment, the particulate carriers are biodegradable, hydrophobic or hydrophilic polymeric microspheres/nanoparticles containing surface COOH, anhydride, NH2, SH groups and combination thereof.
In yet another embodiment, the particulate carriers systems are of both monolithic (matrix type, cross-linked) and/or reservoir type (microcapsule and nanocapsule or multi particulate type (particles within the particles) or particles formed by co-addition the ligand VB12 to the polymer i.e., conjugate polymer particulates.
In yet another embodiment, the particulate carriers include polysaccharide polymers viz., starch and their derivatives, pectin, Amylose, guar gum and their derivatives, dextran of different molecular weights and their derivatives, chitosan and their derivatives, chondriotan sulphate and their derivatives and finally other natural and semi synthetic derivatives of polysaccharides and such polymers thereof.
In yet another embodiment, the cross linking agents for polysaccharides include epichlorohydrin, POCl3, borax (guar gum), aldehydes (e.g., glutaraldehyde) and other cross linking agents thereof which cross links polysaccharide polymers to give particulate carriers.
In yet another embodiment, less hydrophilic to hydrophobic particulate carriers include biodegradable polymers of poly(methylmethacrylate), poly(hydroxybutyrate), polylactide(coglycolic acid), poly(anhydride) microspheres of (fumaric acid: sebacic acid and poly fulmaric acid) and poly (lactide co-glycolide), fatty acylated particulates (hydrophilic core surrounded by fatty acyl layer), LDL carriers, multiparticulate systems (polymeric particles encapsulated in polymers of different hydrophilicity or hydrophobicity) and finally different compositions of these polymers.
In yet another embodiment, the particulate carriers also include natural protein polymers such as albumin, gelatin, semi synthetic or peptide based synthetic polymers and derivatives thereof.
In yet another embodiment, the cross-linking agents such as terephthloyl chloride, glutaraldehyde and such cross linkers which cross-links protein polymers to give particulate carriers.
In yet another embodiment, the particulates, which are formed by polymerization of monomers such as glutaraldehyde to poly (glutaraldehyde), and such particles formed by polymerization of monomers and their coupling to VB12.
In yet another embodiment, the size of the system for the systemic delivery of drugs, proteins and vaccines are ranging from few nanometers to 10 xcexcm or more.
In yet another embodiment, these particulate systems which are modified or activated on the surface to suit VB12 linkage and/or complexation.
In yet another embodiment, the particulate system is surface modified with VB12, and includes coupling of VB12 to biodegradable particulate carriers.
In yet another embodiment, coupling of VB12 derivative and particulates include both biodegradable and non biodegradable bonds with and without spacers in between.
In yet another embodiment, coupling between VB12 derivative and particulate carriers or surface modified/activated particulates is by amide bond with carbodiimides such as 1-ethyl 3-3dimethyl amino propyl carbodiimide (EDAC) or 1,1xe2x80x2 carbonyl diimidazole (CDI) or N,Nxe2x80x2 diisopropyl carbodiimide (DIPC) and other peptide coupling agents or N-hydroxy succinmide activated coupling (NHS) or periodate coupling or glutaraldehyde activated coupling or CNBR mediated coupling or acid halide induced amide coupling or by the use of hydrophilic spacer ethylene glycol bis(succinimidyl succinate) (EGS) or hydrophobic spacers disuccininidyl suberate (DSS) or via a thiol cleavable spacer with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and any such conjugates which retains IF affinity for VB12.
In yet another embodiment, the coupling involves physical adsorptive type or complexation.
In yet another embodiment, the drugs, therapeutic peptides/proteins and vaccines are entrapped within the highly dense VB12 coupled biodegradable particulate carriers.
In yet another embodiment, the loading step of the bioactive agent in question is in the preformed particulate carrier conjugate or within the carrier during the preparation of the particulate spheres or during the coupling between the particles and VB12 derivative.
In yet another embodiment, the bioactives to be delivered are 1) physically entrapped with in the particles before/after/or during the conjugation or 2) adsorbed or 3) covalently coupled or 4) by ionic interaction or 5) complexation and any other methods there off to load VB12 particulate systems.
In yet another embodiment, the use of surfactants, aggregation minimizers, protease inhibitors and permeation enhancers as adjuvants in the formulations.
In yet another embodiment, the delivery systems are co-administered with exogenous intrinsic factor (IF) of VB12, especially for IF deficient species.
In yet another embodiment, the said delivery systems are formulated into dosage forms suitable for oral delivery.
In yet another embodiment, the oral dosage forms include solutions, suspensions, gels, pastes, elixirs, viscous colloidal dispersions, tablets, capsules and/or oral control release types and finally any such delivery forms for oral route.
Oral delivery of peptide and proteins has proven an elusive target for the pharmaceutical industry over the past few decades. In the present invention a model therapeutic protein, insulin and vaccine hepatitis xe2x80x98Bxe2x80x99 vaccine were selected as model protein. The presence of various groups on VB12 derivatives were assigned by comparing IR spectra with that of native VB12. The xe2x80x98exe2x80x99 carboxylate of VB12, which has highest affinity for IF affinity, was separated and covalently linked to various spacers to give various VB12 derivatives (FIG. 2). Various VB12 particulate systems of different sizes were prepared and their surface was modified by various means to suit VB12 conjugation (sizes were illustrated in the text). These VB12 coupled particulate systems (FIG. 1) were loaded with insulin and hepatitis xe2x80x98Bxe2x80x99 vaccine. It is assumed, even if some of the carrier systems delivers sub therapeutic doses of protein (insulin), its relevance would be adequate to trigger immune response. Consequently the problem is far less complicated for traditional drugs that are exclusively given parenterally (hence model drug was not chosen) and will be loaded at a later stage. For these studies permeation enhancers were not taken along with delivery systems, as the main objective is to study uptake mechanism of this carrier system. It is assumed that the above additives may probably potentiate the efficacy of the carrier system by allowing absorption through other mechanisms. In-vitro enzymatic study showed that insulin was protected against digestive action of intestinal enzymes when entrapped within the spheres, whereas free insulin solutions were completely degraded under the same experimental conditions (FIG. 3). Various nanoparticles VB12 conjugates in the size range of 160-250 nm showed interesting results with model protein insulin. Antidiabetic activities of some representative insulin delivery systems are shown in FIGS. 4-8. These systems showed maximum blood glucose reduction of 48.4xc2x14.1 (% initial) at 5 hours. The onset of action was observed 2 hours after administration for all the delivery systems. Maximum % decrease in glucose level (tmax) was found at the 5th hour for all the systems microparticulate conjugates, which was maximal at the 6th hour. The above results show similar uptake kinetics to that observed for VB12 absorption in the rat, and suggest that the uptake be due to specific VB12 mediated absorption, which takes several hours. During preliminary assessment of the different systems the duration of action was studied for 7 hours for all the systems (n=3 animals), but in later studies the uptake was continued for up to 12 hours (n=5) (FIG. 9). The Antidiabetic activity of plain insulin was estimated following intravenous (IV) and subcutaneous administration (FIG. 10). The pharmacological availability was assessed by the above results. The AUC0-7 values were significantly higher (p less than 0.01) for nanoparticle conjugates and higher size nanoparticle conjugates (p less than 0.05) when compared to AUC0-7 value for i.v. route. With microparticulate conjugates there was little difference in AUC""s when compared to i.v. route but there was reduction in blood glucose levels at 3rd, 4th, 5th and 6th hours whereas i.v. injection of insulin showed no activity at these times. The pharmacological availability of various particulate conjugates was from 5% to as much as 23%. It is expected that microparticle conjugates may give good results with vaccines for mucosal immunity, as dose to trigger immune response will not be more than therapeutic proteins (work with hepatitis xe2x80x98Bxe2x80x99 is under progress. Nanoparticle conjugates containing 10 I.U./kg showed best results. Uptake studies showed that the absorption is a VB12 specific carrier-mediated type (FIG. 9). This carrier system and the various strategies described in the present invention have potential to replace the current need for injectable protein drugs/vaccines and other traditional drugs that are given exclusively by parenteral route. These preliminary findings provide a sound platform for further testing and optimizing a delivery technology for peroral delivery of injectable drugs.
For ease of understanding, the distinction between the instant invention and the prior art is schematically represented in FIG. 11. As shown therein, FIG. 11(A) represents the prior art wherein VB12, a bioactive covalent complex is formulated as solutions or as dispersion or as paste or used as tablets, in appropriate dosage forms, FIG. 11(B) represents the instant invention wherein VB12 or its analogues are covalently coupled to microsphere/nanoparticle surface in which bioactive ingredient is loaded and formulate dosage forms.
For easier understanding of this present invention, schematic representation of earlier patents and the present invention is shown in FIG. 11 wherein the Vitamin B12 is covalently linked to the bioactive materials and administered as a solution, dispersion, paste and tablet forms. In the present invention, bioactive (protein/vaccine or drug) is not directly coupled to Vitamin B12 or its analogues, instead Vitamin B12 or its analogues are covalently coupled to microsphere/nanoparticle surface, in which bioactive ingredient is loaded and formulate dosage forms. The micro and nano particles are made up of biodegradable and pharmaceutically acceptable polymers.
As shown in FIG. 11, D is bioactive material, SP is long chain spacer to retain IF affinity of Vitamin B12 and/or for case of coupling BDC is biodegradable carrier particle (microsphere/nanoparticles) and VB12 is Vitamin B12.